Mechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase.

Aspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth.

Involvement of oxidative stress induction in Na+ toxicity and its relation to the inhibition of a Ca2+ -dependent but calcineurin-independent mechanism in Saccharomyces cerevisiae.

Uridine 5'-hexadecylphosphate (UMPC16) inhibited the growth of Saccharomyces cerevisiae under a hypersaline stress condition with Na+ more strongly than the calcineurin inhibitor cyclosporine A (CsA). Additional Ca2+ supplementation similarly suppressed the inhibitory activities of UMPC16 and CsA on yeast cell growth in a medium with Na+. UMPC16, but not CsA, accelerated mitochondrial reactive oxygen species (ROS) generation in combination with Na+, suggesting its inhibition of a Ca2+ -dependent but calcineurin-independent mechanism for protection against Na+ toxicity.

Role of extracellular UTP in the release of uracil from vasoconstricted hindlimb.

The source and function of elevated uracil release during vasoconstriction in the perfused rat hindlimb was investigated. The possibility that uracil release derived from the breakdown of released vasoactive uridine 5'-triphosphate (UTP) was examined. Exogenous UTP was found to be a potent vasodilator in the perfused rat hindlimb, opposing norepinephrine and angiotensin-induced increases in vasoconstriction and oxygen consumption. UTP was rapidly catabolized by the hindlimb to uridine 5'-monophosphate (UMP), uridine, and uracil, which were all devoid of vasoactivity. UTP was similarly catabolized by incubated rat aorta. Degradation of exogenous UTP by perfused hindlimb or aorta was inhibited by alpha, beta-methylene-adenosine 5'-diphosphate (AMP-CP), an inhibitor of ectonucleotidases. However, AMP-CP did not decrease uracil and uridine output by the hindlimb during angiotensin-mediated vasoconstriction and increased oxygen consumption. In particular, simultaneous infusion of AMP-CP with angiotensin did not increase efflux of UMP. Although exogenous UTP is a potent vasodilator in the perfused rat hindlimb, it appears not to be released intact during vasoconstriction. Hence, extracellular UTP is unlikely to be the precursor of the uracil release.

Vasoconstrictor effects of uridine and its nucleotides and their inhibition by adenosine.

Uridine, uridine monophosphate (UMP) and uridine diphosphate (UDP) increased blood pressure when infused into intact anaesthetized rats and had similar effects on the perfusion pressure in the rat isolated perfused kidney. In an isolated vascular preparation, the everted rat portal vein, uridine was without effect while UMP and UDP caused log dose-related increases in contractile work. Adenosine infused at a dose of 200 nmol/kg per min blocked the response to uridine in the intact rat, converting it to a depressor response at higher doses, and reduced the response to UMP. Uridine may need to be phosphorylated to UMP to act on blood vessels. The two compounds are effective at similar dose ranges and suppress renin secretion in the isolated kidney, while UDP, which is effective at lower doses and stimulates renin secretion, may act by a different mechanism. Adenosine competes for membrane transport with uridine but its inhibition of the effects of UMP is consistent with activity at intracellular sites as well.